Symbiotic microorganisms can promote the growth of legumes by way of biological fixation of nitrogen. More specifically, rhizobiaceae are gram-negative soil bacteria which fix nitrogen and are involved in symbiotic association with these legumes. This symbiotic association between the bacteria and the legume enables the latter to grow in soils having low assimilable nitrogen levels. In return, through photosynthesis, the legume provides the bacteria with the energy it requires to reduce the atmospheric nitrogen into ammonia. This ammonia can then be used by the legume and enters into the nitrogen metabolism. The legume, of the Fabaceae family, forms nodules in which the rhizobia proliferate. The Rhizobiaceae family is in a state of taxonomic flux. It has been reported to comprise four main genera: Rhizobium, Bradyrhizobium, Sinorhizobium and Azorhizobium (U.S. Pat. No. 5,549,718). The symbiotic relationship between nitrogen-fixing bacteria or rhizobia and plants of the Fabaceae family enables the growth of the latter in soils having low levels of available nitrogen, thus reducing the need for nitrogen fertilizers. Since nitrogen fertilizers can significantly increase the cost of crops, and are associated with a number of polluting effects, biological means to stimulate this symbiotic relationship and/or to decrease the use of nitrogen fertilizers is of great importance.
Initial recognition between B. japonicum and soybean involves exchange of molecular signals (Stacey et al, 1995). Legume roots secrete phenolic compounds (Dakora & Philips, 1996; Peters & Verma, 1990), largely from the area of root hair emergence, which act as chemo-attractants to (brady)rhizobia (Nap & Bisseling, 1990), and activate the nod genes. Flavones, isoflavones, and flavanones have been identified as the inducing molecules for (brady)rhizobial chemotaxis and for expression of nod genes, e.g. genistein, daidzein and several related compounds in soybean (Peters & Verma, 1990). These plant-to-bacteria signal compounds cause expression of the bacterial nod (also nol and noe) genes very rapidly (only a few minutes after exposure) and at very low concentrations (10−7 to 10−8 M) (Peters et al., 1986). Generally this is through an interaction with nodD, which activates the common nod genes, although the situation may be more complex, as is the case in B. japonicum, where nodD1, nodD2 and nodVW are involved (Gillette & Elkan 1996; Stacey 1995). Nod genes have been identified in the rhizobia that form nitrogen fixing relationships with numbers of the Fabiaceae family (see U.S. Pat. No. 5,549,718 and references therein). Recently, the plant-to-bacteria signal molecules have been shown to promote soybean nodulation and nitrogen fixation under cool soil temperatures (CA 2,179,879) and increase the final soybean grain yield on average of 10% in the field and up to 40% under certain conditions.
Among the products of the nod genes induced by the plant phenolic signal molecules are various enzymes involved in the synthesis of a series of lipo chitooligosaccharides (LCOs) (Spaink, 1995; Stacey, 1995). These newly synthesized LCOs act as bacterium-to-plant signals, inducing expression of many of the early nodulin genes (Long, 1989). This results in root hair deformation (including curling), cortical cell division leading to initiation of nodule meristems, secretion of additional nod gene inducers, and initiation of infection threads (Verma, 1992). These bacterium-to-plant signals exert a powerful influence over the plant genome and, when added in the absence of the bacteria, can induce the formation of root nodules (Truchet et al., 1991). Thus, the bacteria-to-plant signals can, without the bacteria, induce all the gene activity for nodule organogenesis (Denarie et al., 1996; Heidstra & Bisseling, 1996). Moreover, the above-mentioned activities induced by LCOs can be produced by concentrations as low as 10−14 M (Stokkermans et al. 1995). The mutual exchange of signals between the bacteria and the plant are essential for the symbiotic interaction. Rhizobia mutants unable to synthesize LCOs will not form nodules. Analysis of the B. japonicum nod genes indicates that ability to induce soybean nodulation requires at least: 1) a basic tetrameric Nod factor requiring only nodABC genes or 2) a pentameric LCO(C18:1, C16:0 or C16: fatty acid and a methyl-fucose at the reducing end, sometimes acetylated) requiring nodABCZ genes (Stokkermans et al. 1995).
When added to the appropriate legume, LCOs can cause the induction of nodule meristems (Denarie et al., 1996), and therefore cell division activity. One previous publication has shown that LCOs can induce cell cycle activities in a carrot embryogenesis system at levels as low as 10−14 M (De Jong et al. 1993).
A chemical structure of lipo chitooligosaccharides, also termed “symbiotic Nod signals” or “Nod factor”, has been described in U.S. Pat. Nos. 5,549,718 and 5,175,149. These Nod factors have the properties of a lectin ligand or lipo-oligosaccharide substances which can be purified from bacteria or synthesized or produced by genetic engineering.
The relationship between environmental variables, such as low root zone temperature (RZT) and pH, and the interplay of molecular signals has only recently become a subject of investigation. For example, some soybean genotypes have less synthesis abilities for isoflavones under cool soil temperature, whereas a higher isoflavone concentration is needed to turn on the nod genes of B. japonicum (Zhang and Smith 1995 and 1997). The plant-to-bacteria signal molecules (i.e. isoflavones) have been shown, among other things, to overcome the negative effect of low temperature on the early events of symbiotic nitrogen fixation (Canadian application number 2,179,879).
While the effects of plant-to-bacteria signal molecules (i.e. isoflavones) on nodulation, nitrogen fixation, growth and protein yield of legumes, such as soybean, and on bacteria-to-plant signal molecules (LCOs) on nodulation and nitrogen fixation in legumes have been described under certain conditions, the effect of the bacteria-to-plant signal molecules on the growth of non-legumes is unknown. In fact, the role of such bacteria-to-plant signal molecules on non-legumes has never been assessed. In addition, the effect of LCOs on processes other than nodulation of legumes has yet to be studied.
There thus remains a need to assess the effect of LCOs on seed germination, seedling emergence and/or growth of plants in general and especially of non-legume plants.
The present invention seeks to meet these and other needs.
The present description refers to a number of documents, the contents of which are herein incorporated by reference in their entirety.